High performance liquid chromatographic method for determination of doxycycline in human plasma and its application in pharmacokinetic studies

ABSTRACT

An accurate, sensitive and reproducible high performance liquid chromatographic [HPLC] method for the quantitation of doxycycline in plasma has been developed and validated. The drug and the internal standard were eluted from a micro-Bondapak C [18] column [3.9 mm x 150 mm, I.D., 5 micro m particle size] at room temperature with a mobile phase consisting of acetonitrile and water [2872,% v/v]. The flow rate was 0.8 ml/min. A UV detector set at 346 nm was used to monitor the effluent. Each analysis required no longer than 6 min. Quantitation was achieved by measurement of the peak area ratio of the drug to the internal standard. The limit of detection was 10.0 ng/ml and the limit of quantification of doxycycline in plasma was 0.10 micro g/ml. The standard curve ranged from 0.1 to 2.5 micro g/ml. The intraday coefficient of variation [C.V.,%] ranged from 1.444 to 3.016%, and interday [C.V.,%] from 1.572 to 2.705% at four different concentrations. The relative recoveries ranged from 98.43 to 105.13% and the absolute recoveries ranged from 54.08 to 62.56% at four different concentrations. Stability studies showed that doxycycline is stable for at least 4 weeks in plasma after freezing at - 20 °C. The method was applied for the determination of the pharmacokinetic parameters of doxycycline after oral administration of 100 mg capsules of two commercially available formulations to 6 human volunteers